Phenolphthalein-mono-phosphate derivatives



United States Patent ice ,atemed 1 3 The compounds of this invention maybe produced 3,331,862 according to the following schematic diagram:PHENOLPHTHALEIN-MQNO-PHOSPHATE OH OH 0 OH DERIVATIVES I I II I Edward J.Merrill, Whippany, and Arthur L. Babson, 5

Morris Plains, N.J., assignors to Warner-Lambert Pharmaceutical Company,Morris Plains, N.J., a corporam tion of Delaware 6 No Drawing. FiledJune 5, 1964, Ser. No. 373,082

3 Claims. Cl. 260396) 10 O This invention relates to novelmono-phosphate salts =0 COOCHB of phenolphthalein having the structuralformula:

I O O- Phenolphthaleln 0 o-i I H \O /P-O CHsOHrCN I I HO dicyelohexylX[catlon] earbodiimide 3 011 OH w r x i C O O- OH OCH2CHzCN l 1. 011-, AD.

2. 11+ i 0 wherein the cation X is a mono, di, or tri-valent metallic OCOOOHa ion such as, for example, sodium, potassium, calcium, barium,copper, aluminum or iron, or a protonated or- 3: I III ganic amine suchas ammonium and n is an integer. The 0 IV total of the combined cationvalences and those of the phenolphthalein phosphate moiety are equal toeach other. Thus, for example, when the cation is a monol(e.g.NaoH)valent sodium ion, the mono-phosphate derivative of O O O-phenolphthalein 1s tri-sodium phenolphthalem-mono-phos- 4 0 0 I phate.

This invention also includes within its scope a novel H 0 process forthe production of the above compounds as T well as intermediatesobtained during their synthesis.

The compounds of this invention are particularly useful as substratesfor the determination of phosphatase enzymes in a procedure such as, forexample, the deter- I mination of alkaline phosphatase in blood serum,because the amount of phenolphthalein released by enzymatic hydrolysisof the ester bears a linear relationship to the enzyme concentration.This relationship is not observed in the case of the hydrolysis of theknown COO- I In the first step of the reaction above, phenolphthaleinphenolphthalem dlphosphate Smce two phosphoryl ram is converted to itsmethyl ester according to the process cals must first be removed beforeany free phenolphthaldescribed by Green and Kino J soc Chem and Ind einis formed. Thus, when utilizing the latter as the sub- (London) 27 41908 Esseiltiaily henolpt'lthalein strate the free phenolphthaleinformed cannot be easily allowed to rect with absolute methanol in thepresence related to the enzyme concentration. A further advantage ofsulfuric acid in an atmosphae of nitrogen to give the of employing thenew and novel Phenoiphthaiein mono corresponding methyl ester II. Theester II is then con- Piiosphate deficit-bed above is thatproportionately more densed with fi-cyanoethyl phosphate anddicyclohexylfree phenolphthalein is released with this substrate for iiid in dry pyridine to give the intermediate any given amount of y afactor which makes this compound III. This condensation may be eifectedat amsubstrate much more sensitive and therefore much more bienttemperature, for example at a temperature of from accurate in themeasurement of low but clinically signifi- 20 to 30 C. over a period of5 days.

cant levels of phosphatase activity. Compound III may be converted tothe free phenolphthalein mono-phosphate by reaction first with a basesuch as 1 N sodium hydroxide followed byreaction with a strong acid suchas 6 N hydrochloric acid.

To form the desired salts of phenolphthalein monophosphate, the latteris treated with compounds such as sodium or potassium hydroxide, and thereaction product is recovered in the form of a hydrated salt.

In order to further illustrate the practice of this invention, thefollowing examples are given:

Example 1 To a magnetically stirred solution of 8.0 g. (25 .mM.)phenolphthalein in 80 cc. of absolute methanol, under a stream ofnitrogen, is added 54.4 cc. of concentrated sulphuric acid at such arate as to maintain gentle refluxing A fter complete addition, gaseoushydrogen chloride is bubbled through the reaction mixture while allowingit to reflux gently for one 'hour. The cooled reaction mixture and 140cc. of concentrated ammonium hydroxide is simultaneously added to 500cc. ether, which is cooled and stirred in an acetone-Dry Ice bath tokeep the temperature below 25 C. The organic phase is washed with 2 cc.cold aqueous ammonium hydroxide, dried with MgSO and stripped to give ared tacky semi-solid. Trituration of this solid repeatedly with 10 cc.portions of anhydrous ether eventually gives a solid which is discarded.The ether extracts are combined and stripped to give 5.2 g. (62.5%) ofmethyl phenolphthalein as an orange flufiy solid. This material is keptin a vacuum desiccator over KOH.

Example 2 tion stripped to dryness in vacuo. The residue is dissolved in25 cc. dry pyridine and 6.18 g. (30 mM.)

7 dicyclohexyl carbodiimide added in 25 cc. dry pyridine.

An almost immediate precipitate appears. After 5 days at roomtemperature the tightly .stoppered reaction is opened, 5 cc. H O addedand after 3 hours at 20 .to 30 C.

is filtered. The filtrate is stripped to dryness and the resulting oildissolved in 50 cc. 1 N NaOH, filtered and then refluxed for minutes.Solution is cooled and extracted with 3X 15 cc. CHCl The aqueous phaseis clarified by filtration and the cooled filtrate acidified by theaddition of 15 cc. 6 N HCl. The supernate is decanted from a viscous oilwhich is the crude phenolphthaleinmono-phosphate. The crudephenolphthalein-monophosphate is dissolved in sufficient 1 N NaOH togive a;

strongly alkaline solution. Anequal volume of MeOH is added, followed by5 volumes of ether. Trituration of the oily precipitate with 3x 10 cc.acetonitrile gives a yellow solid which is further purified byreprecipitation from a methanol solution by addition of two volumes 7mono-phosphate is obtained as a yellow solid which reddens at ca. C. andthen gradually blackens but does not melt below 400 C.

Analysis for C H O PNa -1 /2H O: Calcd: P. 6.31%; Na, 14.04%; H O,5.50%. Found: P, 6.37%; Na, 14.82%; H O, 5.3%. 7

IR (Nujol): 720, 750, 830, 880, 990, 1150 (broad), 1235, 1500, 1535,1585, 1600, 2850, 2900.

It is to be understood that the foregoing detailed description is givenmerely by way of illustration and that many variations may be madetherein without departing from the spirit of our invention.

Having described our invention, what we desired to secure by LettersPatent is: i

1. A compound of the formula:

Qua..- r

X (cat 1011) I COOCH:

References Cited UNITED STATES PATENTS 5/1961 Walsh 260982 5/1964 Plaut260978 OTHER REFERENCES Henningson et al.: Chemical Abstracts, vol. 53,column Huggins et al.:

J. Biol. Chem., vol. 159, pp. 399-402 ALEX MAZEL, Primary Examiner. J.A. NARCAVAGE, Assistant Examiner.

1. A COMPOUND OF THE FORMULA: